Takae Kiyama

Eomesodermin, a target gene of Pou4f2, is required for retinal ganglion cell and optic nerve development in the mouse

The mechanisms regulating retinal ganglion cell (RGC) development are crucial for retinogenesis and for the establishment of normal vision. However, these mechanisms are only vaguely understood. RGCs are the first neuronal lineage to segregate from pluripotent progenitors in the developing retina. As output neurons, RGCs display developmental features very distinct from those of the other retinal cell types. To better understand RGC development, we have previously constructed a gene regulatory network featuring a hierarchical cascade of transcription factors that ultimately controls the expression of downstream effector genes. This has revealed the existence of a Pou domain transcription factor, Pou4f2, that occupies a key node in the RGC gene regulatory network and that is essential for RGC differentiation. However, little is known about the genes that connect upstream regulatory genes, such as Pou4f2 with downstream effector genes responsible for RGC differentiation. The purpose of this study was to characterize the retinal function of eomesodermin (Eomes), a T-box transcription factor with previously unsuspected roles in retinogenesis. We show that Eomes is expressed in developing RGCs and is a mediator of Pou4f2 function. Pou4f2 directly regulates Eomes expression through a cis-regulatory element within a conserved retinal enhancer. Deleting Eomes in the developing retina causes defects reminiscent of those in Pou4f2-/- retinas. Moreover, myelin ensheathment in the optic nerves of Eomes-/- embryos is severely impaired, suggesting that Eomes regulates this process. We conclude that Eomes is a crucial regulator positioned immediately downstream of Pou4f2 and is required for RGC differentiation and optic nerve development.

Overlapping spatiotemporal patterns of regulatory gene expression are required for neuronal progenitors to specify retinal ganglion cell fate.

Retinal progenitor cells (RPCs) are programmed early in development to acquire the competence for specifying the seven retinal cell types. Acquiring competence is a complex spatiotemporal process that is still only vaguely understood. Here, our objective was to more fully understand the mechanisms by which RPCs become competent for specifying a retinal ganglion cell (RGC) fate. RGCs are the first retinal cell type to differentiate and their abnormal development leads to apoptosis and optic nerve degeneration. Previous work demonstrated that the paired domain factor Pax6 and the bHLH factor Atoh7 are required for RPCs to specify RGCs. RGC commitment is marked by the expression of the Pou domain factor Pou4f2 and the Lim domain factor Isl1. We show that three RPC subpopulations can specify RGCs: Atoh7-expressing RPCs, Neurod1-expressing RPCs, and Atoh7-Neurod1-expressing RPCs. All three RPC subpopulations were highly interspersed throughout retinal development, although each subpopulation maintained a distinct temporal pattern. Most, but not all, RPCs from each subpopulation were postmitotic. Atoh7-Neurod1 double knockout mice were generated and double-mutant retinas revealed an unexpected role for Neurod1 in specifying RGC fate. We conclude that RPCs have a complex regulatory gene expression program in which they acquire competence using highly integrated mechanisms.

Epitope‐tagging Math5 and Pou4f2: New tools to study retinal ganglion cell development in the mouse

Although immunological detection of proteins is used extensively in retinal development, studies are often impeded because antibodies against crucial proteins cannot be generated or are not readily available. Here, we overcome these limitations by constructing genetically engineered alleles for Math5 and Pou4f2, two genes required for retinal ganglion cell (RGC) development. Sequences encoding a peptide epitope from haemagglutinin (HA) were added to Math5 or Pou4f2 in frame to generate Math5HA and Pou4f2HA alleles. We demonstrate that the tagged alleles recapitulated the wild‐type expression patterns of the two genes, and that the tags did not interfere with the function of the cognate proteins. In addition, by co‐staining, we found that Math5 and Pou4f2 were transiently co‐expressed in newly born RGCs, unequivocally demonstrating that Pou4f2 is immediately downstream of Math5 in RGC formation. The epitope‐tagged alleles provide new and useful tools for analyzing gene regulatory networks underlying RGC development. Developmental Dynamics 238:2309–2317, 2009.

Structure, expression, and transcriptional regulation of the Strongylocentrotus franciscanus spec gene family encoding intracellular calcium-binding proteins

The mechanisms by which gene expression patterns emerge during evolution are poorly understood. The sea urchin spec genes offer a useful means to investigate evolutionary mechanisms. Genes of the spec family from Strongylocentrotus purpuratus and Lytechinus pictus have identical patterns of aboral ectoderm-specific expression but exhibit species-specific differences in copy number, genomic structure, temporal expression, and cis-regulatory architecture. Here, we identify spec genes from a phylogenetic intermediate, Strongylocentrotus franciscanus, to gain insight into the evolution of the spec gene family and its transcriptional regulation. We identified two spec genes in the S. franciscanus genome, sfspec1a and sfspec1b, that were orthologous to spec1 from S. purpuratus. sfspec1b transcripts began to accumulate at the blastula stage and became progressively more abundant; this was reminiscent of spec expression in L. pictus but different from that in S. purpuratus. As expected, sfspec1b expression was restricted to aboral ectoderm cells. The six-exon structure of the sfspec1b genomic locus was identical to that of the S. purpuratus spec genes and was bounded by two repeat–spacer–repeat (RSR) repetitive sequence elements, which are conserved features of S. purpuratus spec genes and function as transcriptional enhancers. The enhancer activity of the sfspec1b RSRs was comparable to that of their S. purpuratus counterparts, although the placement and orientation of crucial cis-regulatory elements within the RSRs differed. We discovered a spec gene in S. franciscanus that was only distantly related to other spec genes but was highly conserved in S. purpuratus. Unexpectedly, this gene was expressed exclusively in endoderm lineages. Our results show that the evolution of spec cis-regulatory elements is highly dynamic and that substantial alterations can occur when maintaining or grossly modifying gene expression patterns.

SpGataE, a Strongylocentrotus purpuratus ortholog of mammalian Gata4/5/6: protein expression, interaction with putative target gene spec2a, and identification of friend of Gata factor SpFog1

In the sea urchin Strongylocentrotus purpuatus, SpGataE, an ortholog of the vertebrate zinc-finger transcription factors Gata4/5/6, occupies a key position in the gene regulatory network for endomesoderm specification. We have posited that in addition to regulating gene activity required for endomesoderm specification, SpGataE also represses the expression of the aboral ectoderm-specific spec2a gene in endomesoderm territories. Although the expression pattern of spgatae and its role in endomesoderm specification have been described in considerable detail, little is known about SpGataE protein accumulation and its interactions with target genes and coregulatory factors. Our purpose here was to gain further insight into the mechanisms by which SpGataE functions as a transcriptional regulator. To achieve this, we generated an anti-SpGataE antibody to determine the spatiotemporal expression pattern of SpGataE protein and establish whether it plays a role in repressing spec2a by binding to gata cis-regulatory elements within the endogenous spec2a enhancer. Because Gata proteins often associate with friend of Gata (Fog) coregulators, we identified an S. purpuratus fog ortholog, spfog1, and showed that SpGataE and SpFog1 physically interacted. Spfog1 transcripts were maximal by early blastula stage but continued thereafter to be expressed at low levels. Knockdown of spfog1 using antisense morpholino oligonucleotides did not produce notable effects on endomesoderm specification or spec2a enhancer activity, suggesting that SpGataE exerts these functions independently of SpFog1. In addition to providing new information on Gata and Fog proteins in sea urchins, the anti-SpGataE antibody developed here should be a useful reagent for future analysis of SpGataE function.

Intragenomic evolution of a transcriptional enhancer in the genome of Strongylocentrotus purpuratus.

General principles for how genomic regulatory elements evolve to alter patterns of gene expression remain vague. The purpose of this study was to gain insights into the evolution of genomic regulatory elements by investigating the unique features of a transcriptional enhancer that directs Spec2a gene expression in Strongylocentrotus purpuratus. The Spec2a enhancer is embedded in a repetitive sequence family interspersed throughout the genome. We surveyed the genome and identified 274 of these sequences. They displayed a continuum of sequence divergence defining high and low divergence classes. Alignment of 52 most related to the Spec2a sequence revealed a complex pattern of rearrangements, insertions and deletions, and base-pair changes. A distance tree for the 52 sequences was constructed and correlated with enhancer activity. Unexpectedly, we found a wide range of activities. Notably, repetitive sequences lacking essential cis-elements found in the Spec2a enhancer still had strong activity. We identified short, conserved motifs within the repetitive sequences that may represent novel cis-regulatory elements. Many repetitive sequences with enhancer activity were found nearby genes, suggesting that they regulate gene expression. The results show that the repetitive sequences are rapidly evolving in the S. purpuratus genome and may serve as a renewable pool of transcriptional enhancers.